Differential identification of Mannheimia haemolytica genotypes 1 and 2 using colorimetric loop-mediated isothermal amplification

Objective Mannheimia haemolytica is the primary bacterial pathogen associated with bovine respiratory disease complex (BRDC). While M. haemolytica has been subdivided into 12 capsular serotypes (ST), ST1, ST2 and ST6 are commonly isolated from cattle. More recently, M. haemolytica strains isolated from North American cattle have been classified into genotypes 1 (ST2) and 2 (ST1 and ST6). Of the two genotypes, genotype 1 strains are frequently isolated from healthy animals whereas, genotype 2 strains are predominantly isolated from BRDC animals. However, isolation of both genotypes from pneumonic lung samples can complicate diagnosis. Therefore, the aim of this study was to develop a colorimetric loop-mediated isothermal amplification (LAMP) assay to differentiate M. haemolytica genotypes. Results The genotype specificity of the LAMP was tested using purified genomic DNA from 22 M. haemolytica strains (10 genotype 1, 12 genotype 2) and strains from four related Pasteurellaceae species; Bibersteinia trehalosi, Mannheimia glucosida, Pasteurella multocida, and Histophilus somni. Genotype 1 (adhesin pseudogene B1) specific-LAMP reactions amplified DNA only from genotype 1 strains while genotype 2 (adhesin G) reactions amplified DNA only from genotype 2 strains. The overall detection sensitivity and specificity of the newly developed colorimetric LAMP assay for each genotype were 100%. The limits of detection of two LAMP assays were 1–100 target gene copies per reaction. LAMP primers designed in this study may help the differential identification of M. haemolytica genotypes 1 and 2. Supplementary Information The online version contains supplementary material available at 10.1186/s13104-023-06272-8.


Introduction
Mannheimia haemolytica (formerly Pasteurella haemolytica) is a Gram negative, opportunistic pathogen that primarily resides as a commensal in the nasopharynx and tonsil regions of the upper respiratory tracts of healthy cattle and other ruminants [1]. M. haemolytica is the primary bacterial pathogen associated with bovine respiratory disease complex (BRDC), commonly known as shipping fever, causing extensive economic losses to the beef and dairy cattle industries in the United States and worldwide [2][3][4]. When the host's immune system is compromised, M. haemolytica can descend to the lower respiratory tract with a concomitant development of pneumonia [5]. BRDC is an acute respiratory infection characterized by the development of necrotizing, fibrinous-, broncho-or pleuro-pneumonia often associated with death, particularly in recently weaned beef calves shortly after entry to feedlots [5].
M. haemolytica can be classified into 12 capsular serotypes (ST) based on rapid plate agglutination or indirect hemagglutination tests using anti-capsular sera [6][7][8]. M. haemolytica ST2 is commonly isolated from healthy animals while ST1 and ST6 are largely isolated from lungs of animals affected with BRDC [1,5]. However, isolation of all three serotypes from samples collected from animals suffering from BRDC can occur and complicate diagnosis [1]. Furthermore, Angen et al., (1999) suggested that serotyping does not always represent a reliable method to identify M. haemolytica or Mannheimia glucosida isolates [6]. Recently, M. haemolytica isolated from North American cattle were classified into two genotypes, 1 and 2, based on multiple SNP allele differences and the presence or absence of several genes, such as outer membrane proteins, a peptidase S6, a ligand-gated channel, an autotransporter outer membrane beta-barrel domaincontaining protein, a porin, and three different trimeric autotransporter adhesins that were specific to genotype 2 as their homologs were either pseudogenes or completely absent in genotype 1 [9,10]. M. haemolytica genotype 1 is primarily represented by ST2 strains while genotype 2 is primarily represented by ST1 and ST6 strains [9,10]. Notomi et al., (2000) developed a novel nucleic acid amplification method, LAMP, that amplifies DNA rapidly with high specificity under isothermal conditions [11]. The LAMP reaction uses a specific DNA polymerase with a strand displacement activity along with four primers which recognize six specific target sequences on template DNA and amplifies them at isothermal conditions [11]. The LAMP reaction can be accelerated by the addition of two loop primers [12]. Since 2000, the LAMP method has emerged as an important and versatile diagnostic technique to detect various pathogens [13]. Multiple detection methods have been used to determine LAMP amplicons such as agarose gel with DNA-binding dyes, turbidity detection with turbidity meter, real-time fluorescence detection with fluorescent dyes, and colorimetric detection with phenol red or hydroxynaphthol blue [11,12,14].
Although capsular serotyping is still the gold standard to identify M. haemolytica serotypes, serotyping is not always accurately identify M. haemolytica or M. glucosida isolates [6]. Furthermore, the lack of access to capsular-specific sera for many laboratories can further limit serotyping capabilities [6]. In addition to serotyping, molecular tools such as conventional PCR, real-time PCR and pulsed-field gel electrophoresis are available to identify M. haemolytica strains, as are a MALDI-TOF assay and culture phenotyping characterizations [15][16][17][18]. Recently, a LAMP assay was developed to identify BRDC pathogens, however, the primer sets described for M. haemolytica appear to be only species-specific and is unable to discriminate serotypes or genotypes [19]. Therefore, the goal of this study was to develop a LAMPbased assay to distinguish M. haemolytica genotype 1 from genotype 2 strains.

Bacterial strains, serotyping, and genomic DNA purification
A total of 22 M. haemolytica strains (genotype 1 = 10 and genotype 2 = 12; samples were collected from BRDC animals from the states of Kansas, Kentucky, Missouri, and Tennessee in 2013) and four related Pasteurellaceae species Bibersteinia trehalosi (n = 1), Mannheimia glucosida (n = 1), Pasteurella multocida (n = 1), and Histophilus somni (n = 1) were used in this study. Most of the M. haemolytica strains used in this study were molecularly serotyped by PCR in a previous study [9] and the remaining non-serotyped strains used here were serotyped by rapid plate agglutination test with serotype-specific rabbit antisera generated against ST1 and ST6 as described previously [8]. Bacterial isolates were grown in trypticase soy agar supplemented with 5% sheep blood (Becton, and Dickinson Co., Sparks, MD) at 37 °C in a humidified atmosphere of 7.5% CO 2 for 16 to 48 h. Genomic DNA of each strain was purified using DNeasy Blood & Tissue Kit as per manufacturer's instructions (Qiagen Inc, Valencia, CA). DNA concentration was determined by measuring the absorbance at 260 nm wavelength using a spectrophotometer.

Design of genotype specific LAMP primers
M. haemolytica genotype 1 adhesin pseudogene B1 (GenBank accession no. CP017495.1, locus tag BG548_01640), genotype 2 adhesin G (GenBank accession no. CP017538.1, locus tag BG586_06285), and leukotoxin D (lktD; GenBank accession no. CP005972.1, locus tag F382_07400) nucleotide sequences were used to design LAMP primers. All the primers were designed using NEB online LAMP Primer Design Tool (https:// lamp. neb. com). Details of primers are shown in Additional file 2: Table S1. They also were checked against the adhesin pseudogene B1 sequences of 36 genotype 1 M. haemolytica strains and the adhesin G sequences of 45 genotype 2 M. haemolytica strains, respectively, with complete genomes in GenBank for variation within primer sites with none detected (Additional file 2). M. haemolytica rsmL specific LAMP primer set used in this study was described elsewhere [19]. All the primers were synthesized by Integrated DNA Technologies (IDT Inc., Coralville, IA).

Colorimetric LAMP assay
Colorimetric LAMP reactions were performed using a pH-based WarmStart ® colorimetric LAMP 2 × master mix (with UDG) and a non-pH-based WarmStart ® multipurpose LAMP/RT-LAMP 2 × master mix (with UDG) containing Bst 2.0 DNA Polymerase (NEB Inc., Ipswich, MA). LAMP reactions were carried out using clear PCR 8-tubes strips in a 25 µl final volume containing 12.5 µl 2 × master mix, 2.5 µl 10 × primer mix (2 µm F3 and B3, 16 µm FIP and BIP, and 4 µm LF and LB, Additional file 2: Table S1), 1 µl purified genomic DNA (5 ng) and 9 µl nuclease-free water. A final concentration of 120 µm hydroxynaphthol blue (CAS No. 63451-34-4; Santa Cruz Biotechnology Inc., Dallas, TX) was added to the non-pH-based LAMP master mix as described previously [14]. Tubes were placed in a thermocycler and incubated at 65 °C for 60 min. A real-time fluorescence detection was also performed with a multi-purpose LAMP kit (LAMP fluorescent dye provided with the kit) using QuantStudio 5 Real-Time PCR system in MicroAmp Optical 96-well reaction plates (ThermoFisher Scientific, Waltham, MA). Briefly, LAMP reactions were carried out using Optical 96-well plates in a 25 µl final volume containing 12.5 µl of WarmStart ® multi-purpose LAMP/RT-LAMP 2 × master mix, 2.5 µl of 10 × LAMP primer mix, 0.5 µl of 50 × fluorescent dye (provided with the kit), 1 µl of genomic DNA, and 8.5 µl nuclease-free water. Plates were incubated at 65 °C for 60 min and fluorescence readings (SYBR Green I filter) were recorded every 30 s. To further confirm the colorimetric results, LAMP products were electrophoresed on 1% (w/v) agarose gels and stained with SYBR-Safe DNA gel stain.

Sensitivity of LAMP assay
Sensitivity analysis of LAMP primers was performed using multi-purpose LAMP master mix supplemented with hydroxynaphthol blue. Briefly, LAMP reactions were carried out using clear PCR 8-tubes strips in a 25 µl final volume containing 12.5 µl of WarmStart ® multi-purpose LAMP/RT-LAMP 2 × master mix, 2.5 µl of 10 × LAMP primer mix, 0.5 µl of 6 mM hydroxynaphthol blue (Santa Cruz Biotechnology Inc., Dallas, TX; CAS 63451-35-4; final concentration = 120 µM), 1 µl of genomic DNA, and 8.5 µl nuclease-free water. The genomic DNAs from one genotype 1 strain and two genotype 2 strains were used here. DNA concentration in nanograms per microliter was converted to copies per microliter using ThermoFisher Scientific 'DNA Copy Number and Dilution Calculator' . Genomic DNA was 10 × serially diluted in nuclease-free water and (10 0 -10 5 copies per microliter) used for LAMP reaction. PCR 8-tubes strips were placed in a thermocycler and incubated at 65 °C for 60 min.

Results and discussion
A variety of nucleic acid amplification methods, such as polymerase chain reaction (PCR), nucleic acid sequencebased amplification (NASBA), self-sustained sequence replication (3SR), and rolling circle amplification (RCA) are available. However, LAMP assay is gaining popularity as a point-of-care and other diagnostic applications due to its simplicity and amplification of DNA/RNA with high specificity within 15-60 min under isothermal conditions without the need for thermocycler [11]. Furthermore, positive reaction can be visually determined without agarose gel electrophoresis when using a pHbased and a non-pH-based colorimetric indicators such as phenol red and hydroxynaphthol blue in the reaction mixture, respectively [11,14]. Since LAMP is a simple, rapid, and sensitive assay, our first goal was to design M. haemolytica species-specific LAMP primers targeting the well-conserved lktD gene of the leukotoxin operon [20]. Purified genomic DNAs from M. haemolytica genotype 2 (strains D153 and D174) and genotype 1 (strain D171) were initially compared with four related Pasteurellaceae species; B. trehalosi, M. glucosida, P. multocida, and H. somni, since they also are opportunistic pathogens that reside as commensals of the upper respiratory tract of cattle [21,22]. The colorimetric LAMP with lktD primers showed a positive detection (indicated by a color change from pink to yellow) only with M. haemolytica strains and not with the related Pasteurellaceae species (Fig. 1a, lanes 4-7). Although rsmL LAMP produced positive results for M. haemolytica, it also showed a positive result for M. glucosida (Fig. 1b). The observation of ladder-like banding patterns on agarose gels with the positive LAMP samples including M. glucosida (which was amplified with rsmL but not lktD primers) confirmed the colorimetric LAMP results (Fig. 1c, d). These findings suggested that lktD (but not rsmL LAMP primers) can be used to identify M. haemolytica.
Although multiple LAMP assays are available to detect pathogens relevant to food animals, only one study has been conducted so far to detect BRDC bacterial pathogens [19]. In that study, the M. haemolytica LAMP was species-specific and was unable to discriminate serotypes and genotypes [19]. Furthermore, under our experimental conditions, rsmL LAMP primers also amplified M. glucosida genomic DNA suggesting a lack of specificity of rsmL primers for M. haemolytica. The second goal of this study was to develop LAMP primers to discriminate genotypes 1 and 2. We used the adhesin pseudogene B1 and the adhesin G gene, which have been observed in genotype 1 and genotype 2 strains, respectively [9], to design LAMP primers for genotype discrimination. A positive reaction with hydroxynaphthol blue in the colorimetric LAMP assay was indicated by a color change from violet to sky blue. As expected, adhesin pseudogene B1 primers produced positive LAMP results only with a genotype 1 strain (Fig. 2a) while adhesin G primers produced positive LAMP results only with two genotype 2 strains (Fig. 2b). No amplification (as indicated by no color change) was observed with related Pasteurellaceae species (Fig. 2a, b). The observed ladder-like patterns on agarose gels only in lanes with positive LAMP samples further confirmed the colorimetric LAMP findings (Fig. 2d, e). The lack of adhesin gene amplification with H. somni and P. multocida genomic DNA was expected since neither adhesin pseudogene B1 nor adhesin G were detected in the genomes of both species in a recently completed study [23].
To determine how early amplification was completed, a real-time LAMP assay was performed using a multi-purpose LAMP kit supplemented with a LAMP fluorescent dye. Analysis of real-time data revealed that genotype 2 specific LAMP primers showed increased fluorescence   Fig. 2c). However, increased signal for genotype 1 specific LAMP primers was at ~ 47 cycles (~ 23.5 min) and maximum signal was at ~ 80 cycles (~ 40 min; blue dotted-line, Fig. 2c). Although genotype 2 LAMP primer set has both loop forward and loop backward primers, only one loop primer for genotype 1 could be generated. Therefore, the apparent delay in amplification of genotype 1 might be attributed to the lack of one loop primer. Similar observations have been previously reported for loop primers [12].
To further confirm the genotype-specificity of primers, we performed LAMP assay with nine genotype 1 and ten genotype 2 M. haemolytica strains which were previously characterized for adhesin genes [9]. Based on the previous study, all nine genotype 1 strains used in this study were ST2 [9]. Five of the previously untyped genotype 2 strains were serotyped by rapid plate agglutination test in this study for ST1 and ST6. Two of the five strains were identified as ST1 while remaining three were identified as ST6. Representative colorimetric LAMP results of eight strains for each genotype are shown in shown in Additional file 1: Fig. S1. As predicted, genotype 1 primers were specific to genotype 1 strains while genotype 2 primers were specific to genotype 2 (Additional file 1: Fig.  S1).
Next, we examined the sensitivity of genotype-specific primers by colorimetric LAMP with the purified genomic DNA from one genotype 1 and two genotype 2 strains. Genotype 1 specific primers were able to detect 100 copies per reaction (Fig. 3a) while genotype 2 specific primers were able to detect 1 copy to 10 copies per reaction (Fig. 3b, c). Agarose gel electrophoresis of LAMP products were consistent with colorimetric LAMP findings (Fig. 3d).